Microsomes hydroxylation reactions

Studies reported in this section would appear to confirm that a real potential exists for modifying the ability of plants to detoxify herbicides, and thus change their selectivity profiles, by limiting the activities of their oxidative enzyme machinery. Further developments along these lines, however, will need to be underpinned by a greater fundamental knowledge of the biochemistry of microsomal hydroxylation reactions, in both crop and weed species. 

In a more comprehensive study, Madani et al. (165) quantified CYP2D6 protein in 20 human jejunum and 31 human livers. They found that the median microsomal-specific CYP2D6 content was less than 8% of the hepatic microsomal content (0.85 vs. 12.8 pmol/mg) and that there was extensive interindividual variability in protein content for both tissues. These investigators also characterized the catalytic activity of the same jejunal microsomes toward the recognized CYP2D6 substrate metoprolol and found that a-hydroxylation reaction rate was significantly correlated with CYP2D6 protein content (r = 0.75). 

Microsomal preparations from yeast-elicited cell cultures of chickpea catalyzed the hydroxylation of formononetin and biochanin A to their 2 - and 3 -hydroxy derivatives.43 44 Neither daidzein nor genistein were accepted as substrates. Both hydroxylation reactions seem to be catalyzed by two distinct enzymes, since they exhibited different physicochemical properties and induction kinetics in cell cultures and roots of chickpeas.45 Both 2 - and 3 -hydroxylations of formononetin are prerequisite reactions in the pathway for biosynthesis of the phytoalexins, medicarpin and maackiain in alfalfa and chickpea, respectively8. 

The hydroxylation reaction is directly effected by an enzyme-hemoprotein, monooxigenase, cytochrome P450 containing protocheme IX. The reduction of the enzyme involves flavin reductases and electron carriers, such as adrenodoxin, rubredoxin, and cytochrome b5. Dioxygen, being a weak one-electron oxidant, is activated after the reduction in the enzyme heme coordination sphere. The various forms of cytochrome P450 from liver microsomes and from Pseudomonas putida have a molecular mass of about 49000. One of the subunits of the enzyme from mitochondria of... 

Figure 9.129 Amount of hydroxylated reaction product formed by 0.75 mg of hepatic microsomal protein in 1.0 mL of reaction mixture incubated at 37°C for different lengths of time. 2a, 6jQ, 7a, and 16a-OHT are the hydroxylated metabolites of testosterone A is androstenedione. (From van der Hoeven, 1984.)...

P. H. Cytochrome P4502C9 is the principal catalyst of racemic acenocoumarol hydroxylation reactions in human liver microsomes. Drug Metah. Disp. 2000,... 

The reaction pathway is given in Fig. 33, and the scheme is comparable to the microsomal hydroxylating system. The reducing equivalents are given by NADH and the putidaredoxin. Putidaredoxin is an intermediate compound between the flavoprotein reduced by NADH and cytochrome P-450. 

Storage of frozen rat liver microsomes for 20 or 48 days at - 15°C resulted in a progressive decrease in enzyme activity for the 6/3- and 16a-hydroxylation of testosterone, but la -hydroxylation activity was stable (36). The in vitro addition of 10-4 M chlorthion almost completely inhibited the 16a-hydroxylation of testosterone by rat liver microsomes but only inhibited the 6/3- and 7a-hydroxylation reaction by 31 and 14 percent, respectively (36,37). 

Carbon monoxide inhibited the 6/3-. la-, and 16a-hydroxylation of testosterone by rat liver microsomes to different extents. A C0/02 ratio of 0.5 inhibited the la-, 6/i-, and 16a-hydroxylation reactions by 14%, 25%, and 36%, respectively, and the ratio of C0/02 needed for 50% inhibition of testosterone hydroxylation in the 16a-, 6/3-, and 7a-positions was 0.93, 1.54, and 2.36, respectively (36,48). Studies on the photochemical action spectrum revealed that CO inhibition of the three hydroxylation reactions was maximally reversed by monochromatic light at 450 nm, but there were differences in the shape of the photochemical reactivation spectra for the 6/3-, la-, and 16a-hydroxylation reactions (36,48). The data from our laboratory summarized above and at the First International Symposium on Microsomes and Drug Oxidation in 1968 pointed to multiple cytochromes P450 with different catalytic activities that were under separate regulatory control (36,45,46), and we indicated that the actual number of cytochromes that participate in the multiple hydroxylation reactions must await the solubilization and purification of the microsomal system (36). The use of different inducers of liver microsomal monooxygenases caused selective increases in the concentration of specific cytochromes P450 in fiver microsomes that greatly facilitated the isolation and purification of these hemoproteins. 

System of microsomal MO comprises three main catalytic components. Cytochrome P450 system (reduced flavoprotein oxygen oxidoreductase, EC 1.14.14.1) participate in hydroxylation reactions through processes of oxidoreduction. In oxidative reactions very important is the presence of oxygen, while in reduction reactions electrons are transferred from heme. In some of these reactions enzyme NADPH-P450 reductase (NADPH fcrricytochromc oxidoreduc-... 

At 334 nm NAD(P)H is near its maximum absorption, and Hb02—Hb spectra are in the vicinity of an isosbestic point (eJSP2 - e m = 1.3). Therefore, from measurements at 436 and 334 nm, it is possible to simultaneously determine both the formation or disappearance of NAD(P)H and the oxygen consumption (Figure 7). This procedure permits rapid and accurate estimation of mitochondrial oxidative phosphorylation, or of hydroxylation reactions that occur in liver microsomes... 

Rahimtula, A.D. and P.J. O Brien (1974). Hydroperoxide catalyzed liver microsomal aromatic hydroxylation reactions involving cytochrome P-450. Biochem. Biophys. Res. Commun. 60, 440—447. 

This is the drug metabolising enzyme system of liver microsomes. The hydroxylation reactions can be written ... 

Heme-coupled monooxygenases contain cytochrome P-450. They are present in microsomes and are responsible for many hydroxylation reactions, e.g. llp-hydroxylation of steroids in the adrenal cortex, 2-hydroxylation of estrc ens in the liver the liver system is especially important in the hydroxylation of drugs and xenobiotics, thus rendering them water soluble, capable of conjugation and easily excreiable. A cytochrome P-450 system responsible for the hydroxylation of camphor (a 5-exo-hydroxylase) has been purified from Pseudomonas putida, and named putidaredoxin it contains FAD, an Fc2S2CyS4 center, and a P-4S0 cytochrome substrate hydroxylation is coupled to the oxidation of NADPH. 

Table 1 Microsomal Enzyme Reactions Envisioned as Hydroxylations (I) Aromatic hydroxylation...

On the basis of experiments with protoplasts from epidermal cells of barley leaf sheaths, the proposal was made that the plasmalemma and/or cell wall was the site of the epicuticular wax synthesizing machinery . The observations summarized above are pertinent in this respect. The cer-cqu determined polypeptide contains the enzymatic activity for the final step in the associated pathway consisting of at least the apparent decarboxylation and hydroxylation reactions. The latter would be expected to occur close to the plant surface. This is where the decarbonylation activity which is the final step in hydrocarbon synthesis in peas has been located more specifically in a cutin containing fraction of a microsomal preparation. That the condensing activity of the 0-ketoacyl elongase, which is... 

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